The present invention is in the field of molecular beacon synthesis and use for detection of target sequences, including juxtaposed sequences produced by splicing or cloning.
Molecular beacons (MBs) are oligonucleotides designed for the detection and quantification of target nucleic acids (e.g., target DNAs). The basic principles of molecular beacon mediated target nucleic acid detection is depicted in FIG. 1.
As depicted, 5xe2x80x2 and 3xe2x80x2 termini of the MB collectively comprise a pair of moieties which confers detectable properties of the MB. As shown, one of the termini is attached to a fluorophore and the other is attached to a quencher molecule capable of quenching a fluorescent emission of the fluorophore. For example, one example fluorophore-quencher pair can use a fluorophore such as EDANS or fluorescein, e.g., on the 5xe2x80x2-end and a quencher such as Dabcyl, e.g., on the 3xe2x80x2-end.
When the MB is present free in solution, i.e., not hybridized to a second nucleic acid, the stem of the MB is stabilized by complementary base pairing. This self-complementary pairing results in a xe2x80x9chairpin loopxe2x80x9d structure for the MB in which the fluorophore and the quenching moieties are proximal to one another. In this confirmation, the fluorescent moiety is quenched by the fluorophore.
The loop of the molecular beacon is complementary to a sequence to be detected in the target nucleic acid, such that hybridization of the loop to its complementary sequence in the target forces disassociation of the stem, thereby distancing the fluorophore and quencher from each other. This results in unquenching of the fluorophore, causing an increase in fluorescence of the MB.
Further details regarding standard methods of making and using MBs are well established in the literature and MBs are available from a number of commercial reagent sources. Further details regarding methods of MB manufacture and use are found, e.g., in Leone et al. (1995) xe2x80x9cMolecular beacon probes combined with amplification by NASBA enable homogenous real-time detection of RNA.xe2x80x9d Nucleic Acids Res. 26:2150-2155; Tyagi and Kramer (1996) xe2x80x9cMolecular beacons: probes that fluoresce upon hybridizationxe2x80x9d Nature Biotechnology 14:303-308; Blok and Kramer (1997) xe2x80x9cAmplifiable hybridization probes containing a molecular switchxe2x80x9d Mol Cell Probes 11:187-194; Hsuih et al. (1997) xe2x80x9cNovel, ligation-dependent PCR assay for detection of hepatitis C in serumxe2x80x9d J Clin Microbiol 34:501-507; Kostrikis et al. (1998) xe2x80x9cMolecular beacons: spectral genotyping of human allelesxe2x80x9d Science 279:1228-1229; Sokol et al. (1998) xe2x80x9cReal time detection of DNA:RNA hybridization in living cellsxe2x80x9d Proc. Natl. Acad. Sci. U.S.A. 95:11538-11543; Tyagi et al. (1998) xe2x80x9cMulticolor molecular beacons for allele discriminationxe2x80x9d Nature Biotechnology 16:49-53; Bonnet et al. (1999) xe2x80x9cThermodynamic basis of the chemical specificity of structured DNA probesxe2x80x9d Proc. Natl. Acad. Sci. U.S.A. 96:6171-6176; Fang et al. (1999) xe2x80x9cDesigning a novel molecular beacon for surface-immobilized DNA hybridization studiesxe2x80x9d J. Am. Chem. Soc. 121:2921-2922; Marras et al. (1999) xe2x80x9cMultiplex detection of single-nucleotide variation using molecular beaconsxe2x80x9d Genet. Anal. Biomol. Eng. 14:151-156; and Vet et al. (1999) xe2x80x9cMultiplex detection of four pathogenic retroviruses using molecular beaconsxe2x80x9d Proc. Natl. Acad. Sci. U.S.A. 96:6394-6399. Additional details regarding MB construction and use is found in the patent literature, e.g., U.S. Pat. No. 5,925,517 (Jul. 20, 1999) to Tyagi et al. entitled xe2x80x9cDetectably labeled dual conformation oligonucleotide probes, assays and kits;xe2x80x9d U.S. Pat. No. 6,150,097 to Tyagi et al (Nov. 21, 2000) entitled xe2x80x9cNucleic acid detection probes having non-FRET fluorescence quenching and kits and assays including such probesxe2x80x9d and U.S. Pat. No. 6,037,130 to Tyagi et al (Mar. 14, 2000), entitled xe2x80x9cWavelength-shifting probes and primers and their use in assays and kits.xe2x80x9d
MBs are gaining wide spread acceptance as robust reagents for detecting and quantitating nucleic acids, including in real time (e.g., MBs can be used to detect targets as they are formed). A variety of commercial suppliers produce standard and custom molecular beacons, including Chruachem (chruachem.com), Oswel Research Products Ltd. (UK; oswel.com), Research Genetics (a division of Invitrogen, Huntsville Ala. (resgen.com)), the Midland Certified Reagent Company (Midland, Tex. mcrc.com) and Gorilla Genomics, LLC (Alameda, Calif.). A variety of kits which utilize molecular beacons are also commercially available, such as the Sentinel(trademark) Molecular Beacon Allelic Discrimination Kits from Stratagene (La Jolla, Calif.) and various kits from Eurogentec SA (Belgium, eurogentec.com) and Isogen Bioscience BV (The Netherlands, isogen.com).
Despite such widespread acceptance and commercial development of MBs and related technologies, there remain a number of areas for improvement in the design, manufacture, synthesis, and purification of MBs. For example, in the area of single nucleotide polymorphism (SNP) detection, one typically designs, tests and synthesizes MBs separately for each SNP. This is, of course, inefficient and expensive at several levels. For example, the design and testing process is labor intensive. Additionally, it is difficult to scale the amount of MB actually needed to the synthesis scheme used to make the MB. That is, it can be difficult to scale a synthetic reaction down to produce only as much material as is actually neededxe2x80x94which, with the evolution of modern laboratory systems that run and detect reactions in nanoliter volumes, can be extremely small indeed.
Further in this regard, there are a number of specific difficulties with current synthetic schemes for making MBs. First, MB oligonucleotides require labels on both the 5xe2x80x2 and 3xe2x80x2 ends of the oligonucleotide. Adding 5xe2x80x2 and 3xe2x80x2 labels to oligonucleotides increases their cost dramatically, since specialized CPG (controlled pore glass) supports for solid-phase synthesis are typically used for the 3xe2x80x2 attachment, and specialized phosphoramidites are required for the 5xe2x80x2 attachment. Second, MBs are generally long oligonucleotides (typically greater than 30 nucleotides in length). The longer an oligonucleotide, the lower the percentage of final oligonucleotide product which is full-length, due to the compounding likelihood of synthesis failure at each base. Oligonucleotide purity, therefore, decreases as a function of oligonucleotide length, reducing the effectiveness of the MB and increasing the requirement for purification following synthesis. Indeed, typically, MB oligonucleotides are purified to operate according to specifications. Purification of oligonucleotides that differ by one or a few bases in length is best achieved by polyacrylamide gel electrophoresis (PAGE)-based methods, which are relatively labor intensive and, therefore, expensive. Finally, once designed and synthesized, there is a significant probability that a given MB will be ineffective, due to interfering secondary structure in its own loop region, or interfering secondary structure in the sequence of the target DNA to which the MB hybridizes, which interferes with the hybridization of the MB and target sequence.
The present invention uses modular synthesis strategies to overcome scalability, purification and synthesis issues noted above and to substantially decrease the amount of time needed to design and test MBs. Libraries, kits, devices, ligation mixtures and methods to achieve these goals are provided.
A fuller understanding of the invention will be provided by review of the following.
The present invention uses ligation-based assembly to make MBs. That is, MB components such as the stem, loop, label and label quenching moieties are made separately and then assembled by chemical or enzymatic ligation. Basic approaches include template-based, multiple template based and non-template based ligation assembly reactions. The MBs and components used to make the MBs can comprise nucleic acids, peptide nucleic acids, or both. Most typically, ligation dependent changes in label output are used to monitor ligation of MB components. Methods, devices, ligation mixtures and libraries are provided for high-throughput synthesis and ligation optimization.
Accordingly, the invention comprises methods of making one or more molecular beacon or molecular beacon component (a sub part of a complete molecular beacon). In the methods, a first oligonucleotide or peptide nucleic acid (PNA) corresponding to a first subsequence of a molecular beacon is provided (e.g., by synthesizing the component). At least a second oligonucleotide or PNA corresponding to a second subsequence of a molecular beacon is also provided (as set forth in more detail below, the MB can be made by ligation of 2 or more elements). The first and second oligonucleotides or PNAs are ligated together, thereby forming the molecular beacon (or the molecular beacon component, where the ligation scheme uses more than 2 oligonucleotides to make the MB). One or more additional oligonucleotide or PNA can also be included in the ligation reaction to produce the MB or MB component.
Most typically, the first oligonucleotide or PNA includes a label moiety and the second oligonucleotide includes a label quenching moiety. Common label moieties include those derived from Texas red, terbium chelate, europium cryptate, Fluorescein, IAEDANS, EDANS, BODIPY FL or the like. Common quenching moieties include TRITC (tetrarhodamine isothiocyanate), Allophycocyanin, EDANS, Tetramethylrhodamine, DABCYL, Fluorescein, BODIPY FL, QSY 7 dye or the like.
In a significant aspect, the method optionally includes monitoring a ligation-dependent change in a signal output of the molecular beacon, or of the first or second oligonucleotide or PNA. The ligation-dependent signal output is, e.g., a change in a fluorescence emission at a hybridization temperature that permits intra-molecular hybridization of the molecular beacon, but does not permit inter-molecular hybridization of the molecular beacon. The fluorescence emission change correlates to synthesis of the MB. Detection of this change can be used for a variety of purposes including optimizing one or more reaction parameters to increase yield of the molecular beacon or to improve the efficiency of the ligating step. Similarly, one or more reaction parameters can be optimized to minimize an amount of unligated material remaining following the ligating step.
Detection of the ligation-dependent change (i.e., formation of the MB from separate oligonucleotides) is dependent on the melting and self-annealing of any MB that is actually assembled. Thus, the invention optionally includes using melting and annealing profiles of ligation-dependent emission changes to identify one or more MBs that have an optimized structural component (loop or stem) sequence.
As noted, both template and non-template dependent ligation reactions can be used. For example, the first and second oligonucleotides or PNAs can be aligned on a template nucleic acid prior to said ligating step. The template oligonucleotide can participate in the ligation reaction (thus becoming part of the final MB) or can not participate in the reaction. In this later embodiment, the ends of the oligonucleotide or PNA can be structured to prevent ligation, e.g., in the case of an oligonucleotide by not including phosphate or hydroxyl groups at the terminus of the oligonucleotide. Most commonly, the template nucleic acid is a synthetic single-stranded oligonucleotide, though, e.g., PNAs or cloned nucleic acids can also be used to align MB components in the ligation reaction. Typically, the ligating step is performed via enzymatic ligation, though chemical ligation approaches can also be used. Common ligase enzymes suitable for the ligation reaction include Taq DNA ligase, E. coli DNA ligase, and T4 DNA ligase.
One advantage of the present invention is that purification of the MB from components used to make the MB is simplified due to the substantial difference in size between the MB and the oligos or PNAs used to make the MB. Thus, one aspect of the invention includes purifying the molecular beacon from one or more unligated first or second oligonucleotides or PNAs. Common purification methods include simple purification methods such as HPLC, ion-exchange chromatography or the like.
It will be appreciated from the foregoing that ligation mixtures, e.g., contained in device comprising detectors for monitoring ligation-dependent changes in MB signal output, as well as libraries of ligation components, e.g., used in the methods are also a feature of the invention. For example, ligation mixtures that include a first oligonucleotide or PNA comprising a label moiety, a second oligonucleotide or PNA comprising a quenching moiety that quenches the label moiety when placed proximal or in contact with the label moiety, a third oligonucleotide or PNA that is at least partly complementary to at least a portion of the first or second oligonucleotides, and a ligase are a feature of the invention. The first and second oligonucleotides or PNAs can also be at least partly complementary. Ligation of the first, second and third oligonucleotides or PNAs can result in formation of a molecular beacon, or the third oligo or PNA can simply be a template used in the ligation reaction (of course, additional oligos can be used as MB component elements, or as additional ligation templates). Thus, in one embodiment, ligation of the first and second oligonucleotides or PNAs results in formation of a molecular beacon with the third oligonucleotide providing a template for ligation of the first and second oligonucleotides. In another embodiment, nucleotides of the first and second oligonucleotides form at least a portion of a molecular beacon stem and nucleotides of the third oligonucleotide forms at least portion of a hairpin loop portion of the molecular beacon. These ligation mixtures can be formed in a device having a ligation reaction region, e.g., a micotiter tray, test-tube, cuvette, microfluidic component or other structure configured to receive the ligation reaction.
In the embodiments above, the second oligonucleotide or PNA is typically at least partly complementary to one or more target nucleic acid, e.g., at least partly complementary to one or more single nucleotide polymorphism (SNP). Thus, the MBs made according to the present invention can be used to detect a target nucleic acid such as a SNP, RNA, DNA or the like.
As noted above, a variety of label and quencher elements can be incorporated into the ligation mixture on either the PNA or oligonucleotide, including labels such as Texas red, terbium chelate, europium cryptate, Fluorescein, IAEDANS, EDANS, and BODIPY FL and quenchers such as TRITC (tetrarhodamine isothiocyanate), Allophycocyanin, EDANS, Tetramethylrhodamine, DABCYL, Fluorescein, BODIPY FL, and QSY 7 dyes. Again, ligases that can be used include E. coli ligase, T4 ligase, Taq ligase and other known ligases. Ligation buffers, e.g., selected to facilitate operation of the ligase enzymes can be included as part of the mixture.
Kits comprising ligation mixture components (typically in unmixed form) and kit components (packaging materials, instructions for using the components to produce one or more molecular beacons, or one or more containers (microtiter trays, eppendorf tubes, etc.) for holding the components are also a feature of the invention. Standards for calibrating any MB detection reaction such as standard target sequences, amplification primers for amplifying a target sequence, or the like, can also be included in the kits of the invention.
One additional feature of the invention includes libraries of molecular beacon components. The libraries are designed, e.g., for the rapid synthesis of variants of an MB, e.g., to test variants against one or more target sequences. Such libraries include, e.g., a set of a plurality of hairpin loop oligonucleotides or PNAs, each of the plurality of hairpin loop oligonucleotides or PNAs comprising a subsequence of at least one molecular beacon, the subsequence comprising less than all of the molecular beacon, and at least one label or label quenching oligonucleotide or PNA. The oligonucleotide or PNA comprises at least one label or label quenching moiety, where ligation of at least one hairpin oligonucleotide or PNA and the label or label quenching oligonucleotide or PNA produces a molecular beacon or molecular beacon subsequence.
Most typically, the library is formatted in a gridded array, such as a microtiter tray, to facilitate access to the components of the library. However, any logically accessible arrangement can be used for the library. Thus, for example, individual members types of the hairpin loop oligonucleotides or PNAs are located in wells of the microtiter tray, with the other MB components being added to the wells for ligation to form MBs.
In one aspect, the hairpin loop oligonucleotide or PNA has a label or label quenching moiety, and ligation of the hairpin loop oligonucleotide or PNA to the label or label quenching oligonucleotide or PNA produces a molecular beacon. In another aspect, the library includes both a label oligonucleotide or PNA and a label quenching oligonucleotide or PNA, where ligation of the label oligonucleotide or PNA, the label quenching oligonucleotide or PNA and the hairpin loop oligonucleotide or PNA produces a molecular beacon.
The library can also include essentially any component of the ligation reaction described above, including enzymes, buffers, and the like. The libraries can also be made and used in kit form, e.g., providing the library in conjunction with packaging materials, instructions for using the library to produce one or more molecular beacons, one or more containers for holding one or more components of the library, one or more ligase enzyme, one or more standard target molecule, one or more amplification oligonucleotides, one or more ligation buffer, or the like.
In one embodiment, the invention provides methods for detecting juxtaposition of two or more target subsequences in a target nucleic acid, e.g., as occurs in RNA splicing (or RNA splicing and reverse transcription, as in cDNA production), cloning or the like. In this class of embodiments, a molecular beacon is formed by ligating a first oligonucleotide complementary to a first target subsequence and a second oligonucleotide complementary to a second target subsequence of the target nucleic acid. The resulting molecular beacon is hybridized to the target nucleic acid and a target-specific hybridization of the molecular beacon to the first and second subsequences is detected.
As noted above, a number of modular MB synthesis strategies are set forth herein. These typically include ligating the oligos that form the MB by aligning the oligonucleotides on one or more template and incubating the resulting hybridized set of oligonucleotides with a ligase. A MB used for juxtaposition detection can be formed from more than one oligo, i.e., one or more additional oligonucleotides can be ligated to the first and/or second oligonucleotides. For example, standard stem oligos comprising labels or label quenching moieties can be ligated.